DESCRIPTION: Copper is an essential trace element in both prokaryotic and eukaryotic cells. Its oxidative potential is required for the normal function of over 30 known enzymes in the electron transport, neurotransmitter, connective tissue and free-radical scavenger systems. However, while copper is essential for life, it is highly toxic when present in excess. Therefore, organisms have evolved exact mechanisms for regulating copper homeostasis. Little is known, however, about the mammalian genes that regulate this fine balance. The understanding of this complex process has been greatly enhanced by the cloning of genes causing two genetic disorders, Menkes and Wilson's diseases, in which copper maldistribution is the major metabolic defect. Both have been found to be copper-binding transmembrane ATPases with strong homology to the P-type ATPases of bacteria and to function in cooper transport. During the current project period, the gene (ATP7A;MNK) for Menkes disease was cloned and studies were begun to investigate its structure, function and role in copper homeostasis. Among the findings since identifying the gene, the basic structure of the MNK gene has been determined and found to be very similar to that of the Wilson s disease gene (ATP7B; WND). The MNK protein has been localized to the Golgi network by immunofluorescence. In addition, the homologous mouse gene has been cloned and used to show that the mottled mouse mutants provide valuable animal models for the study of Menkes disease. In this application, studies are proposed to extend characterization of the MNK gene and its protein product and to identify novel related genes involved in copper homeostasis in mammals. Specific aims are: (1) to demonstrate the intracellular localization of the MNK protein by electron microscopy under normal and copper loading conditions; (2) to perform a comparative study of the developmental expression patterns of the MNK and WND genes in normal and mutant mice; (3) to utilize expression constructs of the MNK gene for analysis of structural components involved in expression, copper channeling and protein localization; and (4) to identify and isolate novel related genes involved in copper transport and homeostasis in mammalian cells. These studies will build upon progress made over the past 5 years and will significantly add to the understanding of copper homeostasis in mammals and the role of the MNK gene in this balance and in Menkes disease.